Modulation of transthyretin expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of transthyretin. The compositions comprise oligonucleotides, targeted to nucleic acid encoding transthyretin. Methods of using these compounds for modulation of transthyretin expression and for diagnosis and treatment of diseases and conditions associated with expression of transthyretin are provided.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledIONIS079C5SEQ.txt, created Feb. 15, 2018, which is 38 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of transthyretin. In particular, this invention relatesto antisense compounds, particularly oligonucleotide compounds, which,in preferred embodiments, hybridize with nucleic acid molecules encodingtransthyretin. Such compounds are shown herein to modulate theexpression of transthyretin.

BACKGROUND OF THE INVENTION

Steroid hormones, thyroid hormones, retinoids, and vitamin D are smallhydrophobic molecules that serve as important signaling moleculesthroughout the body. Although all of these molecules are insoluble inwater, they are made soluble for transport in the bloodstream and otherextracellular fluids by binding to specific carrier proteins, from whichthey dissociate before entering a target cell. One such carrier proteinis transthyretin.

Transthyretin (also known as TTR; TTR, prealbumin; prealbumin,thyroxine; PALB; TBPA; HST2651; amyloidosis 1, included;dysprealbuminemic euthyroidal hyperthyroxinemia, included;hyperthytoxinemia, dysprealbuminemic, included; hyperthytoxinemia,dystransthyretinemic, included; 2 5 amyloid polyneuropathy, multipleforms, included; senile systemic amyloidosis, included) is ahomotetrameric transport protein found in the extracellular fluids ofvertebrates (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).

Transthyretin was first identified as the major thyroid hormone carrierin the cerebrospinal fluid (CSF) and in the serum (Palha, Clin Chem LabMed, 2002, 40, 1292-1300; Seibert, I Biol. Chem, 1942, 143, 29-38).Transthyretin was cloned from adult human cDNA libraries and the genewas subsequently mapped to chromosome region 18q11.2-q12.1 (Mita et al.,Biochem Biophys Res Commun, 1984, 124, 558-564; Sparkes et al., HumGenet, 1987, 75, 151-154; Whitehead et al., Mol Biol Med, 1984, 2,411-423).

The liver and the choroid plexus are the primary sites of transthyretinsynthesis in humans (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).

Transthyretin that is synthesized in the liver is secreted into theblood, whereas transthyretin originating in the choroid plexus isdestined for the CSF. In the choroid plexus, transthyretin synthesisrepresents about 20% of total local protein synthesis and as much as 25%of the total CSF protein (Dickson et al., J Biol Chem, 1986, 261,3475-3478). Transthyretin synthesis has also been identified in the yolksac of developing rats(Soprano et al., Proc Natl Acad Sci USA, 1986, 83,7330-7334); the retina, ciliar body and optic nerve regions of bovineand rat eyes (Martone et al., Biochem Biophys Res Commun, 1988, 151,905-912; Ong et al., Biochemistry, 1994, 33, 1835-1842); human andporcine pancreatic islets (Jacobsson et al., J Histochem Cytochem, 1989,37, 31-37) and, in minor amounts, in the stomach, heart, skeletalmuscle, and spleen of rats (Soprano et al., J Biol Chem, 1985, 260,11793-11798).

It is currently believed that transthyretin serves as a hormonereservoir. As demand for thyroid hormone increases, transthyretinincreases the transport and release of hormone to targets such as brain,kidney, and cardiac tissues, thereby ensuring a uniform hormonedistribution within the cells in each of these tissues (Palha, Clin ChemLab Med, 2002, 40, 1292-1300). Transthyretin transports the thyroidhormones triiodothyronine (T₃) and thyroxine (T₄) as well as theretinol/retinol-binding protein complex. A mouse strain deficient intransthyretin is viable and fertile, yet exhibits significantlydepressed levels of serum retinol, retinol-binding protein, and thyroidhormone, confirming transthyretin's role in maintaining normal levels ofthese metabolites in circulating plasma (Episkopou et al., Proc NatlAcad Sci USA, 1993, 90, 2375-2379). In addition to serving as atransport protein, transthyretin has been reported to have a variety ofother functions, including: inhibiting interleukin-1 production inmonocytes and endothelial cells (Borish et al., Inflammation, 1992, 16,471-484); involvement in the metabolism of the environmental pollutantpolyhalogenated biphenyl(Brouwer and van den Berg, Toxicol ApplPharmacol, 1986, 85, 301-312); and binding pterins (Ernstrom et al.,FEBS Lett, 1995, 360, 177-182). Furthermore, in recent years a linkbetween transthyretin and lipoprotein biology has become increasinglyapparent. A fraction of plasma transthyretin circulates in high densitylipoproteins (HDL) through binding to apolipoprotein A-1 (Sousa et al.,J Biol Chem, 2000, 275, 38176-38181), and transthyretin has been shownto proteolytically process apolipoprotein A-1 (Liz et al., J Biol Chem,2004). Furthermore, transthyretin reabsorption by the kidneys ismediated by the lipoprotein receptor megalin (Sousa et al., J Biol Chem,2000, 275, 38176-38181). This reabsorption serves as a means forpreventing hormone loss in urine. Finally, the major site of degradationfor both transthyretin and lipoproteins is the liver. There isconsiderable evidence that hepatic uptake of both transthyretin andlipoproteins is mediated by an as yet unidentified lipoprotein receptor,suggesting a shared degradation pathway (Sousa and Saraiva, J Biol Chem,2001, 276, 14420-14425).

Transthyretin is associated with both local and systemic amyloidosis, adisorder characterized by extracellular systemic deposition of mutatedor wild-type transthyretin as amyloid fibrils (Cornwell et al., BiochemBiophys Res Commun, 1988, 154, 648-653; Saraiva et al., J Clin Invest,1984, 74, 104-119; Yazaki et al., Muscle Nerve, 2003, 28, 438-442),leading to organ dysfunction and death. Senile systemic amyloidosis is asporadic disorder resulting from the extracellular deposition ofwild-type transthyretin fibrils in cardiac and other tissues. Over 80mutations in transthyretin are associated with familial amyloidoticpolyneuropathy and cardiomyopathy. In most of these cases, inheritanceis autosomal dominant (Reixach et al., Proc Natl Acad Sci USA, 2004,101, 2817-2822). Jiang et al (Jiang et al., Proc Natl Acad Sci USA,2001, 98, 14943-14948) demonstrated that the variant with a valine toisoleucine mutation at amino acid 122 (Val122Ile), which is among themost common amyloidogenic mutations worldwide, increases the velocity ofrate-limiting tetramer dissociation, thereby resulting in acceleratedamyloidogenesis. This finding suggests the possibility that treatmentsfor transthyretin-related amyloidoses may include small molecules thatstabilize the tetrameric form (Adamski-Werner et al., J Med Chem, 2004,47, 355-374; Altland and Winter, Neurogenetics, 1999, 2, 183-188). Smallmolecule stabilizers were also shown to be of use in preventing theformation of amyloid fibrils of the wildtype transthyretin (Reixach etal., Proc Natl Acad Sci USA, 2004, 101, 2817-2822). Other commontransthyretin mutations associated with amyloidosis include Val30Met andGlu61Lys. In vitro studies have shown success using ribozymes tospecifically target and inhibit the expression of the Glu61Lys andVal30Met variants (Propsting et al., Biochem Biophys Res Commun, 1999,260, 313-317; Tanaka et al., J Neurol Sci, 2001, 183, 79-84).Single-stranded oligonucleotides have been used both in vitro and invivo to correct single-base mutation (Val30Met) in transthyretin to thewild-type form through targeted recombination (Nakamura et al., GeneTher, 2004). The success of this therapy was limited, however, with geneconversion rates of 11% in vitro and 9% in vivo. These levels are notsufficient for suppression of the variant transthyretin in clinicalterms (Nakamura et al., Gene Ther, 2004).

Thus liver transplantation is currently the only available therapy forfamilial amyloidotic polyneuropathy. However, this therapy is associatedwith several problems, and does not address conditions which are causedby transthyretin variant production in tissues other than liver (Yazakiet al., Muscle Nerve, 2003, 28, 438-442). Consequently, there remains anunmet need for agents capable of effectively modulating transthyretinexpression (Nakamura et al., Gene Ther, 2004, ; Tanaka et al., J NeurolSci, 2001, 183, 79-84).

The PCT publication WO200259621 and the US pre-grant publication20020160394 claim pharmaceutical compositions for treating obesity,comprising an antisense oligonucleotide that hybridizes to apolynucleotide encoding transthyretin and reduces expression of thepolynucleotide. Also claimed is the use of said oligonucleotide in themanufacture of a medicament for treating obesity (Wu, 2002).

The U.S. Pat. 5,744,368 discloses a primer of 22 nucleotides in lengthtargeted to Exon 4 of transthyretin (Goldgaber et al., 1998).

Antisense technology is an effective means of reducing the expression ofspecific gene products and therefore is uniquely useful in a number oftherapeutic, diagnostic, and research applications for the modulation oftransthyretin expression. The present invention provides compositionsand methods for modulating transthyretin expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, especiallynucleic acid and nucleic acid-like oligomers, which are targeted to anucleic acid encoding transthyretin, and which modulate the expressionof transthyretin. Pharmaceutical and other compositions comprising thecompounds of the invention are also provided. Further provided aremethods of screening for modulators of transthyretin and methods ofmodulating the expression of transthyretin in cells, tissues or animalscomprising contacting said cells, tissues or animals with one or more ofthe compounds or compositions of the invention. Methods of treating ananimal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of transthyretin arealso set forth herein. Such methods comprise administering atherapeutically or prophylactically effective amount of one or more ofthe compounds or compositions of the invention to the person in need oftreatment.

DETAILED DESCRIPTION OF THE INVENTION A. Overview of the Invention

The present invention employs antisense compounds, preferablyoligonucleotides and similar species for use in modulating the functionor effect of nucleic acid molecules encoding transthyretin. This isaccomplished by providing oligonucleotides which specifically hybridizewith one or more nucleic acid molecules encoding transthyretin. As usedherein, the terms “target nucleic acid” and “nucleic acid moleculeencoding transthyretin” have been used for convenience to encompass DNAencoding transthyretin, RNA (including pre-mRNA and mRNA or portionsthereof) transcribed from such DNA, and also cDNA derived from such RNA.The hybridization of a compound of this invention with its targetnucleic acid is generally referred to as “antisense”. Consequently, thepreferred mechanism believed to be included in the practice of somepreferred embodiments of the invention is referred to herein as“antisense inhibition.” Such antisense inhibition is typically basedupon hydrogen bonding-based hybridization of oligonucleotide strands orsegments such that at least one strand or segment is cleaved, degraded,or otherwise rendered inoperable. In this regard, it is presentlypreferred to target specific nucleic acid molecules and their functionsfor such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression oftransthyretin. In the context of the present invention, “modulation” and“modulation of expression” mean either an increase (stimulation) or adecrease (inhibition) in the amount or levels of a nucleic acid moleculeencoding the gene, e.g., DNA or RNA. Inhibition is often the preferredform of modulation of expression and mRNA is often a preferred targetnucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position of 3 0hydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). It is preferred that the antisense compounds of thepresent invention comprise at least 70%, or at least 75%, or at least80%, or at least 85% sequence complementarity to a target region withinthe target nucleic acid, more preferably that they comprise at least 90%sequence complementarity and even more preferably comprise at least 95%or at least 99% sequence complementarity to the target region within thetarget nucleic acid sequence to which they are targeted. For example, anantisense compound in which 18 of 20 nucleobases of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleobases may beclustered or interspersed with complementary nucleobases and need not becontiguous to each other or to complementary nucleobases. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome preferred embodiments, homology, sequence identity orcomplementarity, is between about 80% and about 90%. In some preferredembodiments, homology, sequence identity or complementarity, is about90%, about 92%, about 94%, about 95%, about 2 5 96%, about 97%, about98%, about 99% or about 100%.

B. Compounds of the Invention

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other oligomeric compounds which hybridize to atleast a portion of the target nucleic acid. As such, these compounds maybe introduced in the form of single-stranded, double-stranded, circularor hairpin oligomeric compounds and may contain structural elements suchas internal or terminal bulges or loops. Once introduced to a system,the compounds of the invention may elicit the action of one or moreenzymes or structural proteins to effect modification of the targetnucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown thatthe primary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507).The posttranscriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it hasbeen shown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 2002, 295, 694-697).

The antisense compounds of the present invention also include modifiedcompounds in which a different base is present at one or more of thenucleotide positions in the compound. For example, if the firstnucleotide is an adenosine, modified compounds may be produced whichcontain thymidine, guanosine or cytidine at this position. This may bedone at any of the positions of the antisense compound. These compoundsare then tested using the methods described herein to determine theirability to inhibit expression of transthyretin mRNA.

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are a preferred form of the antisense compoundsof this invention, the present invention comprehends other families ofantisense compounds as well, including but not limited tooligonucleotide analogs and mimetics such as those described herein.

The antisense compounds in accordance with this invention preferablycomprise from about 8 to about 80 nucleobases (i.e. from about 8 toabout 80 linked nucleosides). One of ordinary skill in the art willappreciate that the invention embodies compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength.

In one preferred embodiment, the antisense compounds of the inventionare 13 to 50 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 13 to 50 nucleobases inlength, inclusive as detailed above.

In another preferred embodiment, the antisense compounds of theinvention are 15 to 30 nucleobases in length. One having ordinary skillin the art will appreciate that this embodies compounds of 15 to 30nucleobases in length, inclusive as detailed above.

Particularly preferred compounds are oligonucleotides from about 13 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Itis also understood that preferred antisense compounds may be representedby oligonucleotide sequences that comprise at least 8 consecutivenucleobases from an internal portion of the sequence of an illustrativepreferred antisense compound, and may extend in either or bothdirections until the oligonucleotide contains about 8 to about 80nucleobases.

One having skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes transthyretin.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding transthyretin, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). It is also understood that preferred antisense targetsegments may be represented by DNA or RNA sequences that comprise atleast 8 consecutive nucleobases from an internal portion of the sequenceof an illustrative preferred target segment, and may extend in either orboth directions until the oligonucleotide contains about 8 to about 80nucleobases. One having skill in the art armed with the preferred targetsegments illustrated herein will be able, without undue experimentation,to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

The oligomeric antisense compounds may also be targeted to regions ofthe target nucleobase sequence (e.g., such as those disclosed in Example13) comprising nucleobases 1-80, 81-160, 161-240, 241-320, 321-400,401-480, 481-560, 561-640, 641-650, or any combination thereof.

Oligomeric compounds targeted to nucleobases 3880-3899 of SEQ ID NO: 11,or to nucleobases 6-25, 59-78, 91-119, 126-152, 170-189, 197-216,217-236, 232-251, 250-269, 264-297, 323-361, 425-469, 460-532, 532-619of SEQ ID NO: 4 are also suitable embodiments.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of transthyretin. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding transthyretin and which comprise at least an8-nucleobase portion which is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encodingtransthyretin with one or more candidate modulators, and selecting forone or more candidate modulators which decrease or increase theexpression of a nucleic acid molecule encoding transthyretin. Once it isshown that the candidate modulator or modulators are capable ofmodulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding transthyretin, the modulator may then beemployed in further investigative studies of the function oftransthyretin, or for use as a research, diagnostic, or therapeuticagent in accordance with the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocesssing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999,13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The antisense compounds of the present invention can also be applied inthe areas of drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between transthyretin and a disease state, phenotype, orcondition. These methods include detecting or modulating transthyretincomprising contacting a sample, tissue, cell, or organism with thecompounds of the present invention, measuring the nucleic acid orprotein level of transthyretin and/or a related phenotypic or chemicalendpoint at some time after treatment, and optionally comparing themeasured value to a non-treated sample or sample treated with a furthercompound of the invention. These methods can also be performed inparallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. US. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. I Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingtransthyretin. For example, oligonucleotides that are shown to hybridizewith such efficiency and under such conditions as disclosed herein as tobe effective transthyretin inhibitors will also be effective primers orprobes under conditions favoring gene amplification or detection,respectively. These primers and probes are useful in methods requiringthe specific detection of nucleic acid molecules encoding transthyretinand in the amplification of said nucleic acid molecules for detection orfor use in further studies of transthyretin. Hybridization of theantisense oligonucleotides, particularly the primers and probes, of theinvention with a nucleic acid encoding transthyretin can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of transthyretin in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression oftransthyretin is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step 3 0 of administering to theanimal in need of treatment, a therapeutically effective amount of atransthyretin inhibitor. The transthyretin inhibitors of the presentinvention effectively inhibit the activity of the transthyretin proteinor inhibit the expression of the transthyretin protein. In oneembodiment, the activity or expression of transthyretin in an animal isinhibited by about 10%. Preferably, the activity or expression oftransthyretin in an animal is inhibited by about 30%. More preferably,the activity or expression of transthyretin in an animal is inhibited by50% or more. Thus, the oligomeric antisense compounds modulateexpression of transthyretin mRNA by at least 10%, by at least 20%, by atleast 25%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 75%, by at least 80%, by atleast 85%, by at least 90%, by at least 95%, by at least 98%, by atleast 99%, or by 100%.

For example, the reduction of the expression of transthyretin may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. Preferably, the cells contained within saidfluids, tissues or organs being analyzed contain a nucleic acid moleculeencoding transthyretin protein and/or the transthyretin protein itself.

The antisense compounds of the invention can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Use of thecompounds and methods of the invention may also be usefulprophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base sometimesreferred to as a “nucleobase” or simply a “base”. The two most commonclasses of such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turn,the respective ends of this linear polymeric compound can be furtherjoined to form a circular compound, however, linear compounds aregenerally preferred. In addition, linear compounds may have internalnucleobase complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside backbone of the oligonucleotide. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriaminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphos-phonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;

5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones;

alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562;

5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which arecommonly owned with this application, and each of which is hereinincorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred antisense compounds, e.g., oligonucleotide mimetics,both the sugar and the internucleoside linkage (i.e. the backbone), ofthe nucleotide units are replaced with novel groups. The nucleobaseunits are maintained for hybridization with an appropriate targetnucleic acid. One such compound, an oligonucleotide mimetic that hasbeen shown to have excellent hybridization properties, is referred to asa peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂— ] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified antisense compounds may also contain one or more substitutedsugar moieties. Preferred are antisense compounds, preferably antisenseoligonucleotides, comprising one of the following at the 2′ position:OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, alsoknown as 2′—O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′—O—dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′—O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′—O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂), 2′-allyl (2′—CH₂—CH═CH₂), 2′—O-allyl(2′—O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Antisense compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH2—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Antisense compounds may also include nucleobase (often referred to inthe art as heterocyclic base or simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′, 2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2 °C. and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the antisense compounds of the inventioninvolves chemically linking to the antisense compound one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. These moieties or conjugatescan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve uptake, distribution, metabolism or excretion of thecompounds of the present invention. Representative conjugate groups aredisclosed in International Patent Application PCT/US92/09196, filed Oct.23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of whichare incorporated herein by reference. Conjugate moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Antisense compounds of the invention may also be conjugated to activedrug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. Chimeric antisense oligonucleotidesare thus a form of antisense compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically 2 5 acceptable salts of the compounds of theinvention: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Sodium saltshave been shown to be suitable forms of oligonucleotide drugs.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Liposomes and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. Surfactants and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety. One of skill in the art will recognize thatformulations are routinely designed according to their intended use,i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, 2 5 oligonucleotidesmay be complexed to lipids, in particular to cationic lipids. Preferredfatty acids and esters, pharmaceutically acceptable salts thereof, andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. applications Ser. No. 09/108,673 (filed Jul. 1, 1998),Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filedFeb. 8, 2002, each of which is incorporated herein by reference in theirentirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Optimum dosages may vary depending on the relative potencyof individual oligonucleotides, and can generally be estimated based onEC₅₀s found to be effective in in vitro and in vivo animal models. Ingeneral, dosage is from 0.01 ug to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly, or at desiredintervals. Following successful treatment, it may be desirable to havethe patient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 ug to 100 g per kg of body weight,once or more daily.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same. Each of the references, GenBank accession numbers, andthe like recited in the present application is incorporated herein byreference in its entirety.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

Synthesis of nucleoside phorsphoramidates, including amidites and theirintermediates were prepared as described in U.S. Pat. No. 6,426,220 andpublished PCT WO 02/36743, both of which are incorporated herein byreference.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863. 3′-Deoxy-3′-methylene phosphonate oligonucleotidesare prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050.Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878. Alkylphosphonothioateoligonucleotides are prepared as described in published PCT applicationsPCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO94/02499, respectively). 3′-Deoxy-3′-amino phosphoramidateoligonucleotides are prepared as described in U.S. Pat. No. 5,476,925.Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243. Borano phosphate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. Oligonucleosides:Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289. Formacetal andthioformacetal linked oligonucleosides are prepared as described in U.S.Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linkedoligonucleosides are prepared as described in U.S. Pat. No. 5,223,618.All patents and applications are incorporated herein by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Methods of RNA synthesis are well known in the art (Scaringe,S.

A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al.,J Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. andCaruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S.L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B.J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al.,Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., NucleicAcids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron,1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid, or for diagnostic or therapeuticpurposes.

Example 4 Synthesis of Chimeric Compounds

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 394, asabove. Oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dime thoxytrityl-3′-O-phosphoramidite for the DNAportion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′and 3′ wings. The standard synthesis cycle is modified by incorporatingcoupling steps with increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides-[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides- [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingTransthyretin

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target transthyretin. Thenucleobase sequence of the antisense strand of the duplex comprises atleast an 8-nucleobase portion of an oligonucleotide in Table 1. The endsof the strands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 134) and having a two-nucleobaseoverhang of deoxythymidine(dT) would have the following structure:

  cgagaggcggacgggaccgTT Antisense Strand (SEQ ID NO: 135)  ||||||||||||||||||| TTgctctccgcctgccctggc Complement (SEQ ID NO: 136)

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 134) may be preparedwith blunt ends (no single stranded overhang) as shown:

cgagaggcggacgggaccg Antisense Strand (SEQ ID NO: 134)||||||||||||||||||| gctctccgcctgccctggc Complement (SEQ ID NO: 137)

The RNA duplex can be unimolecular or bimolecular; i.e, the two strandscan be part of a single molecule or may be separate molecules.

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times. Onceprepared, the duplexed antisense compounds are evaluated for theirability to modulate transthyretin expression.

When cells reach 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format using methods known to those skilledin the art.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy using methods knownto those skilled in the art.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. This canbe readily determined by methods routine in the art, for exampleNorthern blot analysis, ribonuclease protection assays, or RT-PCR. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. All cell types were cultured under standardconditions, using methods known to those skilled in the art.

T-24 cells: The human transitional cell bladder carcinoma cell line T-24was obtained from the American Type Culture Collection (ATCC) (Manassas,Va.). For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

A549 cells: The human lung carcinoma cell line A549 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.).

NHDF cells: Human neonatal dermal fibroblast (NHDF) were obtained fromthe Clonetics Corporation (Walkersville, Md.).

HEK cells: Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.).

HepG2 cells: The human hepatoblastoma cell line HepG2 was obtained fromthe American Type Culture Collection (Manassas, Va.). Cells were seededinto 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis. For Northern blotting or otheranalyses, cells may be seeded onto 100 mm or other standard tissueculture plates and treated similarly, using appropriate volumes ofmedium and oligonucleotide.

Treatment with antisense compounds: When cells reached 65-75%confluency, they were treated with oligonucleotide. For cells grown in96-well plates, wells were washed once with 100 μL OPTI-MEM™-1reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Invitrogen Corporation, Carlsbad, Calif.) and the desired concentrationof oligonucleotide. Cells are treated and data are obtained intriplicate. After 4-7 hours of treatment at 37° C., the medium wasreplaced with fresh medium. Cells were harvested 16-24 hours afteroligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-(2-methoxyethyl) gapmers (2′-O-(2-methoxyethyl) nucleotides shownin bold) with a phosphorothioate backbone. For mouse or rat cells thepositive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA,SEQ ID NO: 3, a 2′-O-(2-methoxyethyl) gapmers (2′-O-(2-methoxyethyl)nucleotides shown in bold) with a phosphorothioate backbone which istargeted to both mouse and rat c-raf. The concentration of positivecontrol oligonucleotide that results in 80% inhibition of c-H-ras (forISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA isthen utilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line.

If 80% inhibition is not achieved, the lowest concentration of positivecontrol oligonucleotide that results in 60% inhibition of c-H-ras, JNK2or c-raf mRNA is then utilized as the oligonucleotide screeningconcentration in subsequent experiments for that cell line. If 60%inhibition is not achieved, that particular cell line is deemed asunsuitable for oligonucleotide transfection experiments. Theconcentrations of antisense oligonucleotides used herein are from 50 nMto 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of TransthyretinExpression

Antisense modulation of transthyretin expression can be assayed in avariety of ways known in the art. For example, transthyretin mRNA levelscan be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+ mRNA. The preferred method of RNAanalysis of the present invention is the use of total cellular RNA asdescribed in other examples herein. Methods of RNA isolation are wellknown in the art. Northern blot analysis is also routine in the art.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of transthyretin can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed totransthyretin can be identified and obtained from a variety of sources,such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham,Mich.), or can be prepared via conventional monoclonal or polyclonalantibody generation methods well known in the art.

Example 11 Design of Phenotypic Assays for the Use of TransthyretinInhibitors

Phenotypic assays-Once transthyretin inhibitors have been identified bythe methods disclosed herein, the compounds are further investigated inone or more phenotypic assays, each having measurable endpointspredictive of efficacy in the treatment of a particular disease state orcondition. Phenotypic assays, kits and reagents for their use are wellknown to those skilled in the art and are herein used to investigate therole and/or association of transthyretin in health and disease.Phenotypic assay can be purchased from any one of several commercialvendors.

Example 12 RNA Isolation

Poly(A)+mRNA isolation. Poly(A)+mRNA was isolated according to Miura etal., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+mRNAisolation are routine in the art. Briefly, for cells grown on 96-wellplates, growth medium was removed from the cells and each well waswashed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6,1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex)was added to each well, the plate was gently agitated and then incubatedat room temperature for five minutes. 55 μL of lysate was transferred toOligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. The repetitive pipetting and elution steps maybe automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., ValenciaCalif.). Essentially, after lysing of the cells on the culture plate,the plate is transferred to the robot deck where the pipetting, DNasetreatment and elution steps are carried out.

Example 13 Real-Time Quantitative PCR Analysis of Transthyretin mRNALevels

Quantitation of transthyretin mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including 2 5 in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5× PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5× ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L.J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485nm andemission at 530 nm.

Probes and primers to human transthyretin were designed to hybridize toa human transthyretin sequence, using published sequence information(GenBank accession number BCO20791.1, incorporated herein as SEQ ID NO:4). For human transthyretin the PCR primers were: forward primer:CCCTGCTGAGCCCCTACTC (SEQ ID NO: 5) reverse primer: TCCCTCATTCCTTGGGATTG(SEQ ID NO: 6) and the PCR probe was: FAM-ATTCCACCACGGCTGTCGTCA-TAMRA(SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is thequencher dye. For human GAPDH the PCR primers were: forward primer:GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8) reverse primer: GAAGATGGTGATGGGATTTC(SEQ ID NO: 9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRAis the quencher dye.

Example 14 Northern Blot Analysis of Transthyretin mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human transthyretin, a human transthyretin specific probe wasprepared by PCR using the forward primer CCCTGCTGAGCCCCTACTC (SEQ ID NO:5) and the reverse primer

TCCCTCATTCCTTGGGATTG (SEQ ID NO: 6). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Transthyretin Expression byChimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the humantransthyretin RNA, using published sequences (GenBank accession numberBCO20791.1, incorporated herein as SEQ ID NO: 4, and nucleotides 2009236to 2017289 of the sequence with GenBank accession number NT_010966.10,incorporated herein as SEQ ID NO: 11). The compounds are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the compound binds. Allcompounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P=S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humantransthyretin mRNA levels by quantitative real-time PCR as described inother examples herein. Data are averages from two experiments in whichHepG2 cells were treated with 50 nM of the antisense oligonucleotides ofthe present invention. The positive control ISIS 18078 (SEQ ID NO: 2)was used for this assay. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human transthyretin mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET % ISIS # REGION SEQ ID NO SITE SEQUENCE INHIB SEQ ID NO304237 Exon 1: 11  596 aaacactcaccgtagggcca  6 12 Intron 1 junction304238 Intron 1: 11 1520 caccggtgccctgggtgtag  0 13 Exon 2 junction304239 Intron 2 11 1718 tgagcctctctctaccaagt  0 14 304240 Exon 3: 113880 gtatactcacctctgcatgc 33 15 Intron 3 junction 304241 Intron 3 114039 ttctcagagtgttgtgaatt  0 16 304242 Intron 3 11 6252actctgcataaatacatttt  0 17 304243 Intron 3 11 6967 tcttgttttgcaaattcacg 0 18 304244 Intron 3 11 7192 tgaataccacctatgagaga  0 19 304245 5′UTR  4   6 ctgccaagaatgagtggact 33 20 304246 Start Codon  4   18tgagaagccatcctgccaag  6 21 304247 Start Codon  4   25cagacgatgagaagccatcc  2 22 304248 Coding  4   30 aggagcagacgatgagaagc 1023 304249 Coding  4   59 acacaaataccagtccagca 33 24 304250 Coding  4  60 gacacaaataccagtccagc  0 25 304251 Coding  4   66gcctcagacacaaataccag 14 26 304252 Coding  4   75 gtagggccagcctcagacac  327 304253 Coding  4   86 caccggtgcccgtagggcca 16 28 304254 Coding  4  91 ggattcaccggtgcccgtag 32 29 304255 Coding  4  100aggacacttggattcaccgg 47 30 304256 Coding  4  105 atcagaggacacttggattc  031 304257 Coding  4  110 tgaccatcagaggacacttg 21 32 304258 Coding  4 114 actttgaccatcagaggaca 16 33 304259 Coding  4  126acagcatctagaactttgac 33 34 304260 Coding  4  133 gcctcggacagcatctagaa 3435 304261 Coding  4  146 tgatggcaggactgcctcgg 16 36 304262 Coding  4 170 ttctgaacacatgcacggcc 41 37 304263 Coding  4  185tgtcatcagcagcctttctg  8 38 304264 Coding  4  197 atggctcccaggtgtcatca 3439 304265 Coding  4  203 aggcaaatggctcccaggtg 15 40 304266 Coding  4 210 ttcccagaggcaaatggctc  0 41 304267 Coding  4  217actggttttcccagaggcaa 56 42 304268 Coding  4  222 gactcactggttttcccaga  043 304269 Coding  4  232 cagctctccagactcactgg 44 44 304270 Coding  4 239 gcccatgcagctctccagac 14 45 304271 Coding  4  244tgtgagcccatgcagctctc  3 46 304272 Coding  4  250 ctcagttgtgagcccatgca 3647 304273 Coding  4  257 attcctcctcagttgtgagc 10 48 304274 Coding  4 264 tctacaaattcctcctcagt 34 49 304275 Coding  4  278ctttgtatatcccttctaca 43 50 304276 Coding  4  298 agatttggtgtctatttcca  151 304277 Coding  4  314 caagtgccttccagtaagat 14 52 304278 Coding  4 323 gggagatgccaagtgccttc 53 53 304279 Coding  4  342tctgcatgctcatggaatgg 42 54 304280 Coding  4  353 tgaataccacctctgcatgc  755 304281 Coding  4  360 ttggctgtgaataccacctc  5 56 304282 Coding  4 369 ccggagtcgttggctgtgaa 16 57 304283 Coding  4  401tcagcagggcggcaatggtg  1 58 304284 Coding  4  425 ccgtggtggaataggagtag 6359 304285 Coding  4  427 agccgtggtggaataggagt 53 60 304286 Coding  4 431 cgacagccgtggtggaatag 56 61 304287 Coding  4  438ttggtgacgacagccgtggt 92 62 304288 Coding  4  440 gattggtgacgacagccgtg 7063 304289 Coding  4  442 gggattggtgacgacagccg 73 64 304290 Coding  4 443 tgggattggtgacgacagcc 83 65 304291 Coding  4  449attccttgggattggtgacg 45 66 304292 Stop Codon  4  450cattccttgggattggtgac 27 67 304293 Stop Codon  4  451tcattccttgggattggtga 20 68 304294 Stop Codon  4  460agaagtccctcattccttgg 37 69 304295 3′UTR  4  472 gtccactggaggagaagtcc 4770 304296 3′UTR  4  481 gtccttcaggtccactggag 86 71 304297 3′UTR  4  489catccctcgtccttcaggtc 76 72 304298 3′UTR  4  501 tacatgaaatcccatccctc 5273 304299 3′UTR  4  507 cttggttacatgaaatccca 78 74 304300 3′UTR  4  513aatactcttggttacatgaa 52 75 304301 3′UTR  4  526 ttagtaaaaatggaatactc 2076 304302 3′UTR  4  532 actgctttagtaaaaatgga 57 77 304303 3′UTR  4  539tgaaaacactgctttagtaa 54 78 304304 3′UTR  4  546 tatgaggtgaaaacactgct 4879 304305 3′UTR  4  551 tagcatatgaggtgaaaaca 68 80 304306 3′UTR  4  559ttctaacatagcatatgagg 72 81 304307 3′UTR  4  564 tggacttctaacatagcata 7982 304308 3′UTR  4  572 tctctgcctggacttctaac 75 83 304309 3′UTR  4  578ttattgtctctgcctggact 83 84 304310 3′UTR  4  595 cctttcacaggaatgtttta 4685 304311 3′UTR  4  597 tgcctttcacaggaatgttt 79 86 304312 3′UTR  4  598gtgcctttcacaggaatgtt 80 87 304313 3′UTR  4  600 aagtgcctttcacaggaatg 6888 304314 3′UTR  4  604 tgaaaagtgcctttcacagg  8 89

As shown in Table 1, SEQ ID NOs 15, 20, 24, 29, 30, 34, 35, 37, 39, 42,44, 47, 49, 50, 53, 54, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71,72, 73, 74, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 and 88demonstrated at least 27% inhibition of human transthyretin expressionin this assay and are therefore preferred. More preferred are SEQ ID NOs84, 87, and 86. The target regions to which these preferred sequencesare complementary are herein referred to as “preferred target segments”and are therefore preferred for targeting by compounds of the presentinvention. These preferred target segments are shown in Table 2. Thesesequences are shown to contain thymine (T) but one of skill in the artwill appreciate that thymine (T) is generally replaced by uracil (U) inRNA sequences. The sequences represent the reverse complement of thepreferred antisense compounds shown in Table 1. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds. Also shown in Table 2 is thespecies in which each of the preferred target segments was found.

TABLE 2 Sequence and position of preferred targetsegments identified in transthyretin. TARGET REV COMP SEQ SITE IDSEQ ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN ID NO 220029 11 3880gcatgcagaggtgagtatac 15 H. sapiens  90 220034  4    6agtccactcattcttggcag 20 H. sapiens  91 220038  4   59tgctggactggtatttgtgt 24 H. sapiens  92 220043  4   91ctacgggcaccggtgaatcc 29 H. sapiens  93 220044  4  100ccggtgaatccaagtgtcct 30 H. sapiens  94 220048  4  126gtcaaagttctagatgctgt 34 H. sapiens  95 220049  4  133ttctagatgctgtccgaggc 35 H. sapiens  96 220051  4  170ggccgtgcatgtgttcagaa 37 H. sapiens  97 220053  4  197tgatgacacctgggagccat 39 H. sapiens  98 220056  4  217ttgcctctgggaaaaccagt 42 H. sapiens  99 220058  4  232ccagtgagtctggagagctg 44 H. sapiens 100 220061  4  250tgcatgggctcacaactgag 47 H. sapiens 101 220063  4  264actgaggaggaatttgtaga 49 H. sapiens 102 220064  4  278tgtagaagggatatacaaag 50 H. sapiens 103 220067  4  323gaaggcacttggcatctccc 53 H. sapiens 104 220068  4  342ccattccatgagcatgcaga 54 H. sapiens 105 220073  4  425ctactcctattccaccacgg 59 H. sapiens 106 220074  4  427actcctattccaccacggct 60 H. sapiens 107 220075  4  431ctattccaccacggctgtcg 61 H. sapiens 108 220076  4  438accacggctgtcgtcaccaa 62 H. sapiens 109 220077  4  440cacggctgtcgtcaccaatc 63 H. sapiens 110 220078  4  442cggctgtcgtcaccaatccc 64 H. sapiens 111 220079  4  443ggctgtcgtcaccaatccca 65 H. sapiens 112 220080  4  449cgtcaccaatcccaaggaat 66 H. sapiens 113 220081  4  450gtcaccaatcccaaggaatg 67 H. sapiens 114 220083  4  460ccaaggaatgagggacttct 69 H. sapiens 115 220084  4  472ggacttctcctccagtggac 70 H. sapiens 116 220085  4  481ctccagtggacctgaaggac 71 H. sapiens 117 220086  4  489gacctgaaggacgagggatg 72 H. sapiens 118 220087  4  501gagggatgggatttcatgta 73 H. sapiens 119 220088  4  507tgggatttcatgtaaccaag 74 H. sapiens 120 220089  4  513ttcatgtaaccaagagtatt 75 H. sapiens 121 220091  4  532tccatttttactaaagcagt 77 H. sapiens 122 220092  4  539ttactaaagcagtgttttca 78 H. sapiens 123 220093  4  546agcagtgttttcacctcata 79 H. sapiens 124 220094  4  551tgttttcacctcatatgcta 80 H. sapiens 125 220095  4  559cctcatatgctatgttagaa 81 H. sapiens 126 220096  4  564tatgctatgttagaagtcca 82 H. sapiens 127 220097  4  572gttagaagtccaggcagaga 83 H. sapiens 128 220098  4  578agtccaggcagagacaataa 84 H. sapiens 129 220099  4  595taaaacattcctgtgaaagg 85 H. sapiens 130 220100  4  597aaacattcctgtgaaaggca 86 H. sapiens 131 220101  4  598aacattcctgtgaaaggcac 87 H. sapiens 132 220102  4  600cattcctgtgaaaggcactt 88 H. sapiens 133

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of transthyretin.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 16 Western Blot Analysis of Transthyretin Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to transthyretin isused, with a radiolabeled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGERTM (Molecular Dynamics, Sunnyvale Calif.).

All of the applications, patents and references cited are herebyincorporated herein by reference.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus it is intended that the present invention covermodifications and variations of thi invention. The invention is limitedonly by the claims below.

1.-18. (canceled)
 19. A detection method comprising administering to asubject a modulator of human transthyretin expression, detecting nucleicacid or protein levels of transthyretin in a biological sample from saidsubject, wherein the biological sample is serum, adipose tissue, liver,body fluid, or a tissue or organ of an animal.
 20. The detection methodof claim 19, wherein the nucleic acid of transthyretin is detected bypolymerase chain reaction (PCR).
 21. The detection method of claim 20,wherein detecting the nucleic acid of transthyretin by PCR comprisescontacting a preferred target segment of a nucleic acid moleculeencoding human transthyretin with an oligonucleotide probe, a forwardprimer, and a reverse primer, wherein the probe comprises twofluorescent dyes and the forward primer and reverse primer flank thepreferred target segment of the nucleic acid molecule encoding humantransthyretin.
 22. The detection method of claim 21, wherein the forwardprimer comprises SEQ ID NO:
 5. 23. The detection method of claim 21,wherein the reverse primer comprises SEQ ID NO:
 6. 24. The detectionmethod of claim 21, wherein the probe comprises SEQ ID NO:
 25. Thedetection method of claim 24, wherein the two fluorescent dyes are FAMand TAMRA.
 26. The detection method of claim 19, wherein the nucleicacid or protein level of transthyretin is detected by Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS).
 27. The method of claim 19,wherein the modulator of transthyretin expression comprises an antisensecompound, an antisense oligonucleotide, a ribozyme, a DNAoligonucleotide, an RNA oligonucleotide, or an RNA oligonucleotidehaving at least a portion of said RNA oligonucleotide capable ofhybridizing with RNA to form an oligonucleotide-RNA duplex.
 28. Themethod of claim 20, wherein the antisense compound is single-stranded.29. The method of claim 21, wherein the antisense compound comprises amodified oligonucleotide 20 to 25 nucleobases in length having at leastone modified sugar moiety.
 30. The method of claim 22, wherein themodified oligonucleotide is at least 95% complementary to SEQ ID NO: 4.31. A diagnostic method for identifying a transthyretin disease state ina subject comprising identifying the presence of human transthyretin inthe subject by PCR comprising contacting a preferred target segment of anucleic acid molecule encoding human transthyretin with anoligonucleotide probe, a forward primer, and a reverse primer, whereinthe probe comprises two fluorescent dyes and the forward primer andreverse primer flank the preferred target segment of the nucleic acidmolecule encoding human transthyretin.
 32. The diagnostic method ofclaim 31, wherein the forward primer comprises SEQ ID NO:
 5. 33. Thediagnostic method of claim 31, wherein the reverse primer comprises SEQID NO:
 6. 34. The diagnostic method of claim 31, wherein the probecomprises SEQ ID NO:
 7. 35. The diagnostic method of claim 34, whereinthe two fluorescent dyes are FAM and TAMRA.
 36. The diagnostic method ofclaim 31, wherein the forward primer comprises SEQ ID NO: 5, the reverseprimer comprises SEQ ID NO: 6, and the probe comprises SEQ ID NO:
 7. 37.The diagnostic method of claim 36, wherein the two fluorescent dyes areFAM and TAMRA.
 38. The diagnostic method of claim 31, wherein thepreferred target segment is present in a biological sample from thesubject, wherein the biological sample is serum, adipose tissue, liver,body fluid, or a tissue or organ of the subject.